Heterologous expression of pediocin PA-1 in Pichia pastoris: cloning, expression, characterization, and application in pork bologna preservation.

OBJECTIVE: Pediocin PA-1, an antimicrobial peptide derived from Pediococcus acidilactici PAC1.0, has a potential application as a food preservative thanks to its strong inhibitory activity against the foodborne pathogen L. monocytogenes. This study aimed to produce Pediocin PA-1 from the yeast P. pastoris and evaluate its characteristics. METHODS: Gene encoding Pediocin PA-1 was integrated into P. pastoris X33 genome to establish the strain X33::ped, which could produce and secrete this peptide into culture medium. The antimicrobial activity of Pediocin PA-1 was examined using agar diffusion assay. The stability of pediocin PA-1 was determined based on its remaining antibacterial activity after exposure to proteases and extreme pH and temperatures. The potential use of this bacteriocin in food preservation was demonstrated using the L. monocytogenes infected pork bologna. The anticancer activity of Pediocin PA-1 was also investigated on some cancer cells using MTT assay. RESULTS: We established the yeast P. pastoris X33::ped capable of producing pediocin PA-1 with antimicrobial activity against L. monocytogenes and some other harmful bacteria. Pediocin PA-1 was stable at 100˚C and resistant against pH 1-12 for 1 h, but susceptible to trypsin, α-chymotrypsin, and proteinase K. This peptide could reduce the number of L. monocytogenes in pork bologna by 3.59 log CFU/g after 7 days of storage at 4˚C. Finally, Pediocin PA-1 (25 µg/ml) inhibited the proliferation of A549 and Hela cancer cells. CONCLUSION: We succeeded in producing active Pediocin PA-1 from P. pastoris and demonstrated its potential use in food preservation and pharmaceutical industry.

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli.

BACKGROUND: The recombinant human granulocyte colony stimulating factor conjugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimulating Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. METHODS: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the conjugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. RESULTS: PEGylated GCSF was obtained with high purity (∼97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). CONCLUSION: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

Ethyl acetate extract of Elephantopus mollis Kunth induces apoptosis in human gastric cancer cells.

BACKGROUND: Gastric cancer is one of the most leading causes of cancer death worldwide. Therefore, treatment studies have been being conducted, one of which is screening of novel agents from medicinal herbs. Elephantopus mollis Kunth (EM) belonging to Asteraceae family is a perennial herb with several therapeutic properties including anticancer activity. However, the effect of this species on gastric cancer has not been reported yet. In this study, cytotoxicity of different EM crude extracts was investigated on AGS gastric cancer cell line. Besides, the effects of extract on nuclear morphology, caspase-3 activation, and gene expression were also explored. RESULTS: The results showed that ethyl acetate extract exhibited a remarkably inhibitory ability (IC50 = 27.5 µg/ml) on the growth of AGS cells, while causing less toxicity to normal human fibroblasts. The extract also induced apoptotic deaths in AGS cells as evidenced by cell shrinkage, formation of apoptotic bodies, nuclear fragmentation, caspase-3 activation, and the upregulation of BAK and APAF-1 pro-apoptotic genes related to mitochondrial signaling pathway. Specifically, BAK and APAF-1 mRNA expression levels showed 2.57 and 2.71-fold increases respectively. CONCLUSIONS: The current study not only proved the anti-gastric cancer activity of EM ethyl acetate extract but also proposed its molecular mechanism. The extract could be a potential candidate for further investigation.

Production of recombinant human G-CSF from non-classical inclusion bodies in Escherichia coli.

Recombinant granulocyte colony-stimulating factor (G-CSF) protein produced in Escherichia coli has been widely used for the treatment of neutropenia induced by chemotherapy for decades. In E. coli cells, G-CSF is usually expressed as inactive inclusion bodies, which requires costly and inefficient denaturation and refolding steps to obtain the protein in its active form. However, following the findings of previous studies, we here successfully produced G-CSF in E. coli as non-classical inclusion bodies (ncIBs), which contained likely correctly folded protein. The ncIBs were easily dissolved in 0.2% N-lauroylsarcosine solution and then directly applied to a Ni-NTA affinity chromatography column to get G-CSF with high purity (> 90%). The obtained G-CSF was demonstrated to have a similar bioactivity with the well-known G-CSF containing product Neupogen (Amgen, Switzerland). Our finding clearly verified that the G-CSF production from ncIBs is a feasible approach to improve the yield and lower the cost of G-CSF manufacturing process.